Proper development of neuronal morphology and neural circuits, as well as neuronal maintenance, requires tightly controlled subcellular localization of proteins such as axon guidance cue receptors. Proteins are targeted to specific cell locations by elaborate membrane trafficking and axonal transport systems. Neurons are particularly dependent on high fidelity protein trafficking because of their highly polarized and complex structure. Defects in trafficking and transport underlie multiple human developmental and neurodegenerative diseases, including Alzheimer's disease, Charcot-Marie-Tooth, and Niemann Pick disease. Despite their importance, the mechanisms regulating trafficking processes in neurons are poorly understood, in part due to a paucity of in vivo models in which the machinery and mechanisms of axon transport can be studied. A major challenge to the field and the long term goal of this project is to understand the mechanisms controlling axonal transport and protein localization as neurons develop in their natural environment, where they must integrate multiple extracellular cues. We established a model in which we can image dynamics of neuronal cargo transport, protein localization, and microtubule behavior in the intact zebrafish embryo. Vertebrate sensory neurons extend distinct central and peripheral axons to form the sensory circuit. We found that these axons show distinct responses to axon guidance cues. Moreover, we discovered roles for endosomal trafficking and the kinesin adaptor Calsyntenin-1 (Clstn-1) in differential guidance of sensory axons. In Aim 1 we propose to determine how calsyntenins regulate endosome transport routes to different axon compartments. In Aim 2 we will investigate mechanisms regulating specific localization of receptors for Neurotrophin-3 and Semaphorin3d. We will test the hypothesis that Calsyntenins and another class of kinesin adaptors, the Collapsin response mediator proteins (CRMPs) function to target receptors to specific axon compartments. In Aim 3 we will determine how Clstn-1 and CRMPs organize microtubule polarity and dynamics, processes essential for accurate trafficking and axon growth. Our unique model allows us to connect the molecular events of axonal transport, guidance receptor localization and microtubule organization to specific axon guidance decisions at the time and place they naturally occur. Elucidation of the molecular signals regulating sensory axon growth, guidance, and protein trafficking is critical for understanding neurodegenerative disorders, neuropathic pain disorders and the conditions under which regeneration after axon injury can occur. Our experiments will uncover such mechanisms and thus may help to identify molecular targets for disease treatment.